Immunoelectron microscopic studies of the ultrastructure of myosin filaments in Dictyostelium discoideum.

Abstract

We have examined the characteristics of myosin in situ in Dictyostelium amoebae. By an improved immunofluorescence method, we previously found rod-like structures that contain myosin, which we call "myosin rods", in amoebae (Yumura. S., and Fukui, Y. (1985) Nature, 314: 194-196). Although we prepared samples for electron microscopy using conventional chemical fixation to clarify the ultrastructure of the myosin rods, we could not find any filamentous structures similar to myosin thick filaments. Therefore, we examined the effects of chemical fixatives on the myosin rods in situ by immunofluorescence staining. When cells were fixed in more than 0.05% glutaraldehyde or more than 1% osmium tetroxide at 4 degrees C, the myosin rods disappeared. These effects did not result from loss of the antigenicity, because a monoclonal myosin-specific antibody was able to react with synthetic myosin filaments treated with 0.5% glutaraldehyde or 2% osmium tetroxide. Cells fixed by the procedure used for immunofluorescence staining were post-fixed with permissible concentrations of chemical fixatives and prepared for examination by transmission electron microscopy. We found discrete filaments of about 12 nm thickness between the microfilaments. These filaments were shown to contain myosin by immunoelectron microscopy with an immunogold probe. These filaments were thinner than synthetic myosin thick filaments formed in vitro in the presence of 10 mM MgCl2, but they were similar to those formed in the presence of 2 mM MgCl2, or under nearly physiological ionic conditions. The images after immunofluorescence and immunogold labeling both suggested that these 12-nm-thick filaments in Dictyostelium amoebae were myosin filaments in situ.